Chemokines, also known as "intercrines" and "SIS cytokines", comprise a superfamily of small secreted proteins (approximately 70-100 amino acids and 8-12 kilodaltons in size) which primarily regulate leukocyte migration and activation, and thereby aid in the stimulation and regulation of the immune system. The name "chemokine" is derived from the term chemotactic cytokine, and refers to the ability of these proteins to stimulate chemotaxis of leukocytes. Indeed, chemokines may comprise the main attractants for inflammatory cells into pathological tissues. [See generally, Baggiolini et al., Advances in Immunology, 55:97-179 (1994); Oppenheim, The Chemokines, Lindley et al., eds., pages 183-186, Plenum Press, N.Y. (1993))]. While chemokines are generally secreted by leukocytes, several chemokines are expressed in a multitude of tissues. Baggiolini et al., supra, Table II. Some chemokines also activate or attract a variety of cell types in addition to leukocytes, such as endothelial cells and fibroblasts.
Previously identified chemokines generally exhibit 20-70% amino acid identity to each other and contain four highly-conserved cysteine residues. Based on the relative position of the first two of these cysteine residues, chemokines have been further classified into two subfamilies. In the "C-X-C" or ".alpha." subfamily, encoded by genes localized to human chromosome 4, the first two cysteines are separated by one amino acid. In the "C--C" or ".beta." subfamily, encoded by genes which have been mapped to human chromosome 17, the first two cysteines are adjacent. X-ray crystallography and NMR studies of several chemokines have indicated that, in each family, the first and third cysteines from a first disulfide bridge, and the second and fourth cysteines form a second disulfide bridge, strongly influencing the native conformation of the proteins. In humans alone, nearly ten distinct sequences have been described for each chemokine subfamily. Chemokines of both subfamilies have characteristic leader sequences of twenty to twenty-five amino acids.
The C-X-C chemokines, which include IL-8, GRO.alpha./.beta./.gamma., platelet basic protein, Platelet Factor 4 (PF4), neutrophil-activating peptide-2 (NAP-2), macrophage cheinotactic and activating factor (MCAF), IP-10, and others, share approximately 25% to 60% identity when any two amino acid sequences are compared (except for the GRO.alpha./.beta./.gamma. members, which are 84-88% identical with each other). Most of the subfamily members (excluding IP-10 and Platelet Factor 4) share a common E-L-R tri-peptide motif upstream of the first two cysteine residues. The C-X-C chemokines are generally potent stimulants of neutrophils, causing rapid shape change, chemotaxis, respiratory bursts, and degranulation. Specific truncation of the N-terminal amino acid sequence of certain C-X-C chemokines, including IL-8, is associated with marked increases in activity.
The C--C chemokines, which include Macrophage Inflammatory Proteins MIP-1.alpha. [Nakao et al., Mol. Cell Biol., 10:3646 (1990)] and MIP-1.beta. [Brown et al., J. Immunol., 142:679 (1989)], Monocyte Chemotactic Proteins MCP-1 [Matsushima et al., J. Exp. Med., 169:1485 (1989)], MCP-2 [Van Damme et al., J. Exp. Med., 176:59 (1992) and Chang et al., Int. Immunol., 1:388 (1989)], and MCP-3 [Van Damme et al., supra], RANTES [Schall et al., J. Immunol., 141:1018 (1988)], I-309 [Miller et al., J. Immunol., 143:2907 (1989)], eotaxin [Rothenberg et al., J. Exp. Med., 181:1211-1216 (1995)] and others, share 25% to 70% amino acid identity with each other. The C--C chemokines generally activate monocytes, lymphocytes, basophils and eosinophils, but not neutrophils. Most of the reported C--C chemokines activate monocytes, causing calcium flux and chemotaxis. More selective effects are seen on lymphocytes, for example, T-lymphocytes, which respond most strongly to RANTES.
C--C chemokines can be further subdivided according to structural homologies and similar activities. MIP-1.alpha., MIP-1.beta. and RANTES have closer homology and range of biological activities than the other members of the family. Another subfamily within the C--C chemokine family are the monocyte chemotactic proteins (MCP), which are structurally more similar to each other than to other members of the C--C chemokine family, and which preferentially stimulate monocytes to migrate and respond to inflammatory stimuli.
Studies with deletion and substitution analogs have revealed that the critical receptor binding regions appear to be primarily in the amino-terminal residues of the chemokines, followed by a second region in the loop following the second cysteine. These general requirements for function appear to be common to all chemokines. [Clark-Lewis et al., J. Leukocyte Bio., 57:703 (1995).]
The chemokine receptors are seven-transmembrane-domain rhodopsin-like G protein-coupled receptors. A receptor specific for IL-8 has been cloned by Holmes et al., Science, 253:1278-83 (1991), while a similar receptor (77% identity) which recognizes IL-8, GRO and NAP-2 has been cloned by Murphy and Tiffany, Science, 253:1280-83 (1991). Five of the C--C chemokine receptors have been cloned to date: a C--C chemokine receptor-1 (CCR-1) which recognizes MIP-1.alpha. and RANTES [Neote et al., Cell, 72:415-425 (1993)], a receptor (CCR-4) for MIP-1.alpha., RANTES and MCP-1 [Power et al., J. Biol. Chem., 270:19495-19500 (1995)], an MCP-1 receptor (CCR-2B) [Charo et al., Proc. Nat. Acad. Sci., 91:2752-56 (1994)], an eotaxin receptor (CCR-3) [Combadiere et al., J. Biol. Chem. 270:16491-16494 (1995)], and a receptor (CCR-5) for MIP-1.alpha., MIP-1.beta. and RANTES [Raport et al., J. Biol. Chem., 271:17161-17166 (1996)].
These receptors tend to be multifunctional, and may bind a number of different chemokines. The receptors themselves may play a role in human disease. For example, the Duffy antigen on human red blood cells (also known as the erythrocyte chemokine receptor), which binds avidly to chemokines including IL-8, NAP-2, GRO.alpha., RANTES, MCP-1, is an invasion receptor for a malaria-causing parasite, Plasmodium knowlesi. Two herpesviridae, Herpesvirus saimiri and human cytomegalovirus, also appear to encode functional chemokine receptor homologs. [Ahuja et al., Immunol. Today, 15:281-(1994); Murphy, Ann. Rev. Immunol., 12:593-633 (1994); Horuk, TIPS, 15:159 (1994).]
Because of their pro-inflammatory activities, chemokines are believed to play a role in a wide variety of diseases involving inflammatory tissue destruction, such as rheumatoid arthritis, myocardial infarction, and adult respiratory distress syndrome. The role of a number of chemokines, particularly the C-X-C chemokine IL-8, has been well documented in various pathological conditions. See generally Baggiolini et al., supra, Table VII. For example, several studies have observed high levels of IL-8 in the synovial fluid of inflamed joints of patients suffering from rheumatic diseases, osteoarthritis, and gout. Psoriasis has also been linked to over-production of IL-8.
The role of C--C chemokines in pathological conditions has also been documented. For example, the concentration of MCP-1 is higher in the synovial fluid of patients suffering from rheumatoid arthritis than that of patients suffering from other arthritic diseases. The MCP-1 dependent influx of mononuclear phagocytes may be an important event in the development of idiopathic pulmonary fibrosis. The role of C--C chemokines in the recruitment of monocytes into atherosclerotic areas is currently of intense interest, with enhanced MCP-1 expression having been detected in macrophage-rich arterial wall areas but not in normal arterial tissue. MCPs may also be involved in induction of angiogenesis and tumor growth or metastasis. Expression of MCP-1 in malignant cells has been shown to suppress the ability of such cells to form tumors in vivo. (See U.S. Pat. No. 5,179,078, incorporated herein by reference.)
Other chemokine activities include the ability to inhibit the proliferation of bone marrow progenitor cells. Recombinant MIP-1.alpha., but not MIP-1.beta., has been shown to suppress myelopoiesis of stein and progenitor cells, and appears to be selective in its ability to suppress growth factor-stimulated proliferation of multipotential progenitor cells (colony forming units of granulocyte-erythroid-macrophage-megakaryocytes, CFU-GEMM) and subpopulations of burst-forming units of erythroid (BFU-E) and colony-forming units of granulocytes-macrophages (CFU-GM) progenitor cells. [Broxmeyer et al., Blood, 76:1110-1116 (1990).] These effects are not a cytotoxic effect, but rather a cell cycle arrest. MIP-2.alpha., IL-8, PF4 and MCAF also have been reported to be suppressors of hemopoietic stem/progenitor cell proliferation. [Broxmeyer et al., J. Immunol., 150:3448-3458 (1993); Broxmeyer et al., Ann. Hematol., 71:235-246 (1995).] These chemokines appear to act directly at the level of the myeloid progenitors. Some reports indicate that MIP-1.alpha. has the potential to protect multipotent hematopoietic cells from the cytotoxic effects of chemotherapeutic agents. [Dunlop et al., Blood, 79:2221-2225 (1992) and Lord et al., Blood, 79:2605-2609 (1992).] Clinical trials are reportedly under way for the use of a MIP-1.alpha., analog (designated BB10010, British Biotechnology) as a myeloprotective agent with Cytoxan.RTM. (cyclophosphamide from Bristol-Myers Squibb Oncology).
Recently, there have been several reports that some C--C chemokines, MIP-1.alpha., MIP-1.beta. and RANTES, inhibit human immunodeficiency virus (HIV) production. [Cocchi et al., Science, 270:1811 (1996); Fauci, Nature, 378:561 (1996).] One study has reported that CD4+ lymphocytes of individuals who have been exposed to HIV but remain HIV-negative express very high levels of these C--C chemokines. [Paxton et al., Nature Med., 2:412 (1996).] A potential mechanism for this inhibition has been suggested by the isolation and identification of HIV co-receptors as members of the chemokine receptor families. The CCR-5 receptor which binds RANTES, MIP-1.alpha. and MIP-1.beta. has been identified as the main co-receptor for most macrophage-tropic HIV strains [Deng et al., Nature, 381:661 (1996); Dragic et al., Nature, 381:667 (1996); Alkhatib et al., Science, 272:1955 (1996)]. It has been reported that occasional primary HIV-1 macrophage-tropic strains interact with the CCR-3 and CCR-2B receptors in vitro [Choe et al., Cell, 85:1135 (1996); Doranz et al., Cell, 85:1149 (1996)]. A chemokine receptor designated "Fusin" (now known as the C-X-C chemokine receptor CXCR-4) has been identified as a receptor for T-cell tropic strains of HIV [Feng et al., Science, 272:872 (1996)]. These HIV co-receptors are in the cheinokine receptor families, and appear to be cofactors with CD4 for the fusion and entry of HIV viruses into human target cells.
A need therefore exists for the identification and characterization of additional C--C chemokines, to further elucidate the role of this important family of molecules in pathological conditions and to develop improved treatments for such conditions utilizing chemokine-derived products.
Of interest to the present invention is International Publication No. WO 96/05856 published Feb. 29, 1996, which reports the identification of two chemokines termed human chemokine beta-4 (Ck.beta.-4) and human chemokine beta-10 (Ck.beta.-10) from cDNA libraries derived from human gall bladder and nine week human fetal tissue, respectively. Ck.beta.-4 is very similar in both DNA and amino acid sequence to the Exodus chemokine described herein (the differences being that Ck.beta.-4 has an additional alanine after residue 4 of the mature Exodus chemokine and that the reported deduced leader sequence of Ck.beta.-4 is 24 amino acids, compared to the 22 amino acid leader sequence of Exodus). No biological activities of either chemokine Ck.beta.-4 or Ck.beta.-10 were determined. In particular, the publication does not mention any potential role for these chemokines in the pathogenesis of HIV infection, nor does it specifically describe use of these chemokines for treating myeloproliferative diseases.
Also of interest is the cloning of another C--C chemokine, designated Exodus-2, that appears to be closely related to Exodus/MIP-3.alpha.-LARC, sharing 31% amino acid identity and the same unique Asp-Cys-Cys-Leu motif seen around the first two cysteines. [Hromas et al., J. Immunol., 159:2554-2558 (1997).]
Chemokines of the C--C subfamily have been shown to possess utility in medical imaging, e.g., for imaging the site of infection, inflammation, and other sites having C--C chemokine receptor molecules. See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778, incorporated herein by reference. Such methods involve chemical attachment of a labelling agent (e.g., a radioactive isotope) to the C--C chemokine using art recognized techniques (see, e.g., U.S. Pat. Nos. 4,965,392 and 5,037,630, incorporated herein by reference), administration of the labelled chemokine to a subject in a pharmaceutically acceptable carrier, allowing the labelled chemokine to accumulate at a target site, and imaging the labelled chemokine in vivo at the target site. A need in the art exists for additional new C--C chemokines to increase the available arsenal of medical imaging tools.
More generally, due to the importance of chemokines as mediators of chemotaxis and inflammation, a need exists for the identification and isolation of new members of the chemokine family to facilitate modulation of inflammatory and immune responses. For example, substances that promote the immune response may promote the healing of wounds or the speed of recovery from infectious diseases such as pneumonia. Substances that inhibit the pro-inflammatory effects of chemokines may be useful for treating pathological conditions mediated by inflammation, such as arthritis, Crohn's disease, and other autoimmune diseases.
Additionally, the established correlation between chemokine expression and inflammatory conditions and disease states provides diagnostic and prognostic indications for the use of chemokines, as well as for antibody substances that are specifically immunoreactive with chemokines; a need exists for the identification and isolation of new chemokines to facilitate such diagnostic and prognostic indications.
For all of the aforementioned reasons, a need exists for recombinant methods of production of newly discovered chemokines, which methods facilitate clinical applications involving the chemokines and/or chemokine inhibitors.